Comparison of fluorescent genotyping methods.
Comparison of fluorescent genotyping methods.
复制标题
荧光基因分型方法的比较。
DOI：
10.1006/abio.1998.2778
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Bateman,JB
中科院分区：
文献类型：
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作者：
Johannes,M;Geyer,DD;Berry,R;Walter,NA;Sikela,JM;Bateman,JB
我们比较了使用荧光标记引物和掺入荧光dUTPs（[F] dUTPs）1进行基因型分析，以确定疗效和成本。荧光标记引物的电泳图清晰，等位基因容易识别。由于大小的标准差增加、峰大小减小和峰伪影，[F] dUTP的掺入具有模糊的等位基因鉴定。对于连锁作图，使用荧光标记的引物是优选的方法。使用标记引物的优点包括：（i）更容易对凝胶进行评分，（ii）购买市售标记引物与[F] dUTP相比花费更少，以及（iii）每个泳道易于多路复用几个标记物。[F]尽管如此，dUTP可以与现有的未标记的三核苷酸和四核苷酸引物一起使用，并且比定制标记的引物便宜。材料和方法。为了比较这两种方法，使用标记引物和[F] dUTPs对4个ADC个体的5个基因座进行分析。等位基因大小和频率从基因组数据库获得（1）。PCR反应使用相同的镁浓度和退火温度，用于每个带有标记引物和[F] dUTP的序列标记位点。
We compared the use of fluorescently labeled primers and the incorporation of fluorescent dUTPs ([F] dUTPs) 1 for genotype analyses to determine efficacy and costs. Electropherograms of the fluorescently labeled primers were clear and the alleles easily identified. Incorporation of [F] dUTPs had ambiguous allele identification due to increased standard deviations of size, decreased peak sizes, and peak artifact. For linkage mapping, the use of fluorescently labeled primers is the preferred method. The advantages of using labeled primers include:(i) easier scoring of gels,(ii) less expense with the purchase of commercially available labeled primers versus [F] dUTPs, and (iii) ease of multiplexing several markers per lane.[F] dUTPs, nonetheless, can be used with existing unlabeled trinucleotide and tetranucleotide primers and are less expensive than having labeled primers custom made. Materials and methods. To compare the two methods, five loci were analyzed using labeled primers and [F] dUTPs with four ADC individuals. Allele sizes and frequencies were obtained from the Genome Database (1). PCR reactions used identical magnesium concentrations and annealing temperatures for each sequence-tagged site with both labeled primers and [F] dUTPs.